The present application concerns methods for the identification of functional antisense agents. In particular, the present application is concerned with improved methods for measuring the effectiveness of potential antisense oligonucleotides, via the kinetics of RNA/oligonucleotide hybrid cleavage.
PCT/GB96/02275 describes a method to determine which oligonucleotides, in a library, are active antisense agents, by identification of the sites at which cleavage of a target RNA occurs. The methods of this invention do not identify which of the oligonucleotides in the library of this invention is likely to be the most effective antisense agent.
PCT/GB97/02722 describes a method to identify which regions of an RNA are accessible to hybridisation probes by hybridising short oligonucleotide probes to a target RNA in separate reactions. The sequences of the hybridising probes are compared with the primary sequence of the target RNA to identify probes which hybridise to overlapping sequences in the target RNA. These sequences identify accessible regions in the target RNA. The methods in this application do not determine whether oligonucleotides complementary to an accessible site in a target RNA will mediate cleavage of that RNA by RNAse H.
It is an object of this invention to solve the problems arising from deficiencies in the above prior art methods and to provide a method of determining which oligonucleotides in an array of short oligonucleotide probes are most effective at mediating cleavage of a target RNA by RNAse H. It is also an object of this invention to compare this cleavage data with the primary sequence of the target RNA to identify contiguous sequences within the mRNA that are most accessible to oligonucleotides which can mediate RNAse H cleavage of the target. This will allow antisense agents, of sufficient length to be highly target specific, to be identified using a generic library of oligonucleotide probes that can be used to analyse any target RNA It is a further object of this invention to provide methods of identifying effective antisense agents that meet the earlier objectives without requiring gel electrophoresis or related separation techniques.